Novel pharmaceutical formulations

ABSTRACT

There is provided inter alia a dry powder pharmaceutical formulation for inhalation comprising: (i) 6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide or a pharmaceutically acceptable salt thereof, including all stereoisomers, tautomers and isotopic derivatives thereof and solvates thereof in particulate form as active ingredient; (ii) particulate lactose as carrier; and (iii) a particulate stabilizing agent selected from metal salts of stearic acid such as magnesium stearate and metal salts of stearyl fumarate.

FIELD OF THE INVENTION

The present invention provides novel dry powder pharmaceuticalformulations for inhalation of a compound that inhibits phosphoinositide3-kinases (PI3 kinases), and their use in therapy, especially in thetreatment of inflammatory diseases such as COPD and asthma.

BACKGROUND OF THE INVENTION

Lipid kinases catalyse the phosphorylation of lipids to produce speciesinvolved in the regulation of a wide range of physiological processes,including cellular migration and adhesion. The PI3-kinases are membraneassociated proteins and belong to the class of enzymes which catalysethe phosphorylation of lipids which are themselves associated with cellmembranes. The PI3-kinase delta isozyme (PI3 kinase δ) is one of fourisoforms of type I PI3 kinases responsible for generating various3′-phosphorylated phosphoinositides, that mediate cellular signallingand has been implicated in inflammation, growth factor signalling,malignant transformation and immunity [See Review by Rameh, L. E. andCantley, L. C. J. Biol. Chem., 1999, 274:8347-8350].

The involvement of PI3 kinases in controlling inflammation has beenconfirmed in several models using pan-PI3 kinase inhibitors, such asLY-294002 and wortmannin [Ito, K. et al., J. Pharmacol. Exp. Ther.,2007, 321:1-8]. Recent studies have been conducted using eitherselective PI3 kinase inhibitors or in knock-out mice lacking a specificenzyme isoform. These studies have demonstrated the role of pathwayscontrolled by PI3 kinase enzymes in inflammation. The PI3 kinase δselective inhibitor IC-87114 was found to inhibit airwayshyper-responsiveness, IgE release, pro-inflammatory cytokine expression,inflammatory cell accumulation into the lung and vascular permeabilityin ovalbumin-sensitized, ovalbumin-challenged mice [Lee, K. S. et al.,J. Allergy Clin. Immunol., 2006, 118:403-409 and Lee, K. S. et al.,FASEB J., 2006, 20:455-65]. In addition, IC-87114 lowered neutrophilaccumulation in the lungs of mice and neutrophil function, stimulated byTNFα [Sadhu, C. et al., Biochem. Biophys. Res. Commun., 2003,308:764-9]. The PI3 kinase δ isoform is activated by insulin and othergrowth factors, as well as by G-protein coupled protein signalling andinflammatory cytokines. Recently the PI3 kinase dual δ/γ inhibitorTG100-115 was reported to inhibit pulmonary eosinophilia andinterleukin-13 as well as mucin accumulation and airwayshyperesponsiveness in a murine model, when administered byaerosolisation. The same authors also reported that the compound wasable to inhibit pulmonary neutrophilia elicited by either LPS orcigarette smoke [Doukas, J. et al., J. Pharmacol. Exp. Ther., 2009,328:758-765]

Since it is also activated by oxidative stress, the PI3 kinase δ isoformis likely to be relevant as a target for therapeutic intervention inthose diseases where a high level of oxidative stress is implicated.Downstream mediators of the PI3 kinase signal transduction pathwayinclude Akt (a serine/threonine protein kinase) and the mammalian targetof rapamycin, the enzyme mTOR. Recent work has suggested that activationof PI3 kinase δ, leading to phosphorylation of Akt, is able to induce astate of corticosteroid resistance in otherwise corticosteroid-sensitivecells [To, Y. et al., Am. J. Respir. Crit. Care Med., 2010,182:897-904]. These observations have led to the hypothesis that thissignalling cascade could be one mechanism responsible for thecorticosteroid-insensitivity of inflammation observed in the lungs ofpatients suffering from COPD, as well as those asthmatics who smoke,thereby subjecting their lungs to increased oxidative stress. Indeed,theophylline, a compound used in the treatment of both COPD and asthma,has been suggested to reverse steroid insensitivity through mechanismsinvolving interaction with pathways controlled by PI3 kinase δ [To, Y.et al., Am. J. Respir. Crit. Care Med., 2010, 182:897-904].

International patent application WO2011/048111 discloses a number ofcompounds which are inhibitors of PI3 kinases, particularly PI3 kinaseδ, including6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamidein the free base form which is disclosed therein as Example 83. Thiscompound is also disclosed in WO2012/052753.

The above mentioned compound is referred to herein as “compound offormula (I)” or “compound of formula (I) free base”.

Prior to the applicant's earlier disclosure (WO2011/048111), the PI3kinase inhibitors described to date have typically been intended fororal administration. However, an undesired consequence of this approachis that non-targeted body tissues, especially the liver and the gut, arelikely to be exposed to pharmacologically active concentrations of thedrug. An alternative strategy is to design treatment regimens in whichthe drug is dosed directly to the inflamed organ via topical therapy. Inthe case of controlling inflammation (or providing another therapeuticeffect) in the lungs, this may be achieved by inhalation of the drug,which has the benefit of retaining the drug predominantly in the lungsthereby minimising the risks of systemic toxicity. In order to achieve asustained duration of action an appropriate formulation which generatesa “reservoir” of the active drug may be used.

The compound of formula (I) has, accordingly, been described as beinguseful for topical administration to the lung (see WO2011/048111).

As well as providing affinity for the target organ and sustainedefficacy, a drug for topical administration to the lung via inhalationmust also be formulated so as to provide a predictable dose of the drug,which in turn must have predictable and reproducible properties.Achieving acceptable and reproducible chemical and physical stability ofthe drug in the formulation is a key goal in the product development ofpharmaceutical products for all types of pharmaceutical dosage forms.

For inhalation use, there are 3 main dosage forms—a dry powder inhaler(DPI), a metered dose inhaler (MDI) and an aqueous based nebuliser(hand-held or table-top). However the majority of global sales ofinhalation products are DPIs and thus provide a well-accepted way ofdelivering drugs by inhalation. There are numerous commercialised DPIproducts, such as Flixotide (fluticasone propionate), Advair(fluticasone propionate/salmeterol), Symbicort (budesonide/formoterol),Pulmicort (budesonide), Serevent (salmeterol) and Foradil (formoterol).

Dry powder inhalation formulations typically consist of a blend of drugparticles (size below 10 microns and normally below 5 microns) with adiluent, typically lactose. Since the usual doses required for inhaledtherapies are in the microgram range, the diluent facilitatespharmaceutical processing and dispensing of individual doses e.g. intocapsules or blisters or the metering of doses from a bulk reservoir, forsubsequent administration to the patient. Therefore, typically, the massof diluent (the most common being lactose) may be greater than that ofthe drug substance. In this environment, acceptable formulations of someproducts can be achieved by simply blending the drug with lactose. Otherproducts may require other additional excipients or other processingsteps in order for the product to meet the requirements of regulatoryauthorities.

One such additional excipient is magnesium stearate which is known forimproving certain properties of formulations containing it. Thus, U.S.Pat. No. 7,186,401B2 (Jagotec A G et al.) discloses that the addition ofmagnesium stearate to dry powder formulations for inhalation improvesthe moisture resistance of the formulations and allows a high fineparticle dosage or fine particle fraction to be maintained under humidconditions. WO00/53157 (Chiesi) describes magnesium stearate as alubricant to be employed in dry powder formulations for inhalation whichis capable if increasing the fine particle dose of certain drugs.US2006/0239932 (Monteith) discloses an inhalable solid pharmaceuticalformulation comprising certain active ingredient substances susceptibleto chemical interaction with lactose, lactose and magnesium stearate. Itis disclosed that magnesium stearate inhibits lactose induceddegradation of the active ingredient, presumably via the Maillardreaction which involves the reaction of an amine group on the activeingredient with lactose. US2012/0082727 (Chiesi) discloses a method ofinhibiting or reducing chemical degradation of an active ingredientbearing a group susceptible to hydrolysis selected from the groupconsisting of a carbonate group, a carbamate group and an ester group ina powder formulation for inhalation comprising carrier particles (suchas lactose particles) said method comprising coating at least a portionof the surface of said carrier particles with magnesium stearate.

Thus, there remains a need to provide formulations of selective PI3kinase inhibitors for use in inhalation therapy which have the potentialto provide therapeutic efficacy in asthma, COPD and other inflammatorydiseases of the lungs. In particular, it remains an objective to providea formulation of the compound of formula (I) which has appropriatephysical and chemical stability and other necessary properties forinhalation therapy.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a dry powderpharmaceutical formulation for inhalation comprising:

-   -   (i)        6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)        methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide        (compound of formula (I)) or a pharmaceutically acceptable salt        thereof, including all stereoisomers, tautomers and isotopic        derivatives thereof and solvates thereof in particulate form as        active ingredient;    -   (ii) particulate lactose as carrier; and    -   (iii) a particulate stabilizing agent selected from metal salts        of stearic acid (such as magnesium stearate) and metal salts of        stearyl fumarate

Such a formulation is hereinafter referred to as “a formulation of theinvention”.

As explained in the Examples, formulations of the invention appear tohave good physical stability (as determined by XRPD and IR analysis) andgood chemical stability (as determined by HPLC analysis). Without beinglimited by theory, it appears from the inventors' discoveries that thealkyne group of the compound of formula (I) is susceptible to metalcatalysed oxidative degradation involving hydration of the alkyne. Italso appears from the inventors' discoveries that the pyrimidinone ringof the compound of formula (I) is susceptible to hydrolytic cleavage.Experiments conducted by the inventors have determined that formulationsof the invention containing lactose and a metal salt of stearic acidsuch as magnesium stearate have superior chemical stability thancorresponding formulations not containing a metal salt of stearic acidsuch as magnesium stearate. To the inventors' knowledge it has not beenreported before that a metal salt of stearic acid such as magnesiumstearate can act as a protecting agent against chemical degradation ofalkyne containing compounds (especially in respect of metal catalysedoxidative degradation involving hydration of the alkyne) in dry powderinhalation formulations. To the inventors' knowledge it has also notbeen reported before that a metal salt of stearic acid such as magnesiumstearate can act as a protecting agent against hydrolytic cleavage of adrug substance containing a pyrimidinone ring. The inventors extrapolatethese findings with metal salts of stearic acid to metal salts ofstearyl fumarate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an XRPD pattern acquired on a sample of compound of formula(I) in solid crystalline anhydrous form.

FIG. 2 shows an IR spectrum of a sample of a blend of compound offormula (I) in anhydrous form (micronized) with Lactohale200® andmagnesium stearate.

FIG. 3 shows an XRPD pattern acquired on a sample of a blend of compoundof formula (I) in anhydrous form (micronized) with Lactohale200® andmagnesium stearate.

DETAILED DESCRIPTION OF THE INVENTION Compound of Formula (I) as ActiveIngredient

The compound of formula (I) is a dual PI3K delta PI3K gamma inhibitor,wherein the term inhibitor as employed herein is intended to refer to acompound that reduces (for example by at least 50%) or eliminates thebiological activity of the target protein, for example the PI3K deltaisozyme, in an in vitro enzyme assay. The term delta/gamma inhibitor asemployed herein is intended to refer to the fact that the compoundinhibits, to some degree, both enzyme isoforms although not necessarilyto the same extent. Compound of formula (I) is active in cell basedscreening systems and thereby demonstrates that it possesses suitableproperties for penetrating cells and thereby exert intracellularpharmacological effects.

Generic processes for synthesising the compound of formula (I) aredisclosed in WO2011/048111, the contents of which are incorporated byreference in their entirety, and a method similar to that of Example 1can be employed. See also WO2012/052753, the contents of which areincorporated by reference in their entirety, where a specific method forsynthesising the compound of formula (I) is provided in the Example.

Suitably, compound of formula (I) is protected from light during andafter synthesis e.g. by use of amber glassware or light imperviouspackaging (e.g. foil packaging).

The dry powder pharmaceutical formulation of the present inventioncomprises compound of formula (I) as active ingredient in atherapeutically effective amount. A therapeutically effective amount ofcompound of formula (I) is defined as an amount sufficient, for a givendose or plurality of divided doses, to achieve a therapeuticallymeaningful effect in a subject when administered to said subject in atreatment protocol.

In one embodiment, the dry powder pharmaceutical formulation comprisesfrom about 0.004 wt. % to about 50 wt. % of compound of formula (I)based on weight of the dry powder pharmaceutical formulation and basedon weight of compound of formula (I) as free base; for example fromabout 0.02 wt. % to about 50 wt. %, from about 0.02 wt. % to about 25wt. %, from about 0.02 wt. % to about 20 wt. %, or from about 0.02 wt. %to about 15 wt. %. Preferably, the dry powder pharmaceutical formulationcomprises from about 0.1 wt. % to about 20 wt. % e.g. from about 0.1 wt.% to about 5 wt. % of compound of formula (I) based on the weight of thedry powder pharmaceutical formulation and based on weight of compound offormula (I) as free base.

A pharmaceutical formulation of the invention may contain compound offormula (I) as a single active ingredient. However, the pharmaceuticalformulation may contain further active ingredients. The pharmaceuticalformulation may also be co-administered together with one or more otheractive ingredients (or one or more pharmaceutical formulationscontaining one or more active ingredients). Exemplary further activeingredients are mentioned below.

Compound of formula (I) is prepared in particulate form such that it issuitable for dry powder inhalation. A pharmaceutical formulation of theinvention may typically contain drug particles having a volume mediandiameter (D50) from about 0.5 μm to about 10 μm particularly from about1 μm to about 5 μm.

A suitable method for determining particle size is laser diffraction,e.g. using a Mastersizer 2000S instrument from Malvern Instruments.Instruments are also available from Sympatec. For particle sizedistributions, the median value D50 is the size in microns that splitsthe particle size distribution with half above and half below. Theprimary result obtained from laser diffraction is a volume distribution,therefore D50 is actually Dv50 (median for a volume distribution) and asused herein refers to particle size distributions obtained using laserdiffraction. D10 and D90 values (when used in the context of laserdiffraction, taken to mean Dv10 and Dv90 values) refer to the particlesize wherein 10% of the distribution lies below the D10 value, and 90%of the distribution lies below the D90 value, respectively.

Particles of suitable size for use in a dry powder inhalationformulation may be prepared by any suitable method known to the personskilled in the art. Drug particles of suitable size for inhalation maybe prepared by particle size reduction methods including milling or morepreferably micronization e.g. using a jet mill micronization device(e.g. as manufactured by Hosokawa Alpine). Alternatively, particulatesof suitable size may be produced at the first instance by spray drying,spray freezing, controlled crystallisation approaches e.g. controlledprecipitation, super-critical fluid crystallisation, sonocrystallisationor other suitable crystallisation procedure, for example in a continuouscrystallisation apparatus.

In one embodiment, compound of formula (I) is in free base form, in theform of a pharmaceutically acceptable salt, or in the form of a solvateof either. Suitably compound of formula (I) is in free base form, e.g.in anhydrous form.

Suitably, compound of formula (I), or a pharmaceutically acceptable saltor solvate thereof, is in solid crystalline form.

Pharmaceutically Acceptable Salts of Compound of Formula (I)

In one embodiment there is provided a pharmaceutically acceptable saltof compound of formula (I).

The pharmaceutically acceptable salts as mentioned hereinabove are meantto comprise the therapeutically active non-toxic acid addition saltforms that the compound of formula (I) is able to form. Thesepharmaceutically acceptable acid addition salts conveniently can beobtained by treating the base form with such appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-aminosalicylic, pamoic and the like acids.

Thus, specific examples of salts of compound of formula (I) include theacid additional salts formed with HCl, HBr and p-toluenesulfonic acid.

Solvates

The invention also extends to solvates of compound of formula (I).Examples of solvates include hydrates and hygroscopic products such aschannel hydrates.

Anhydrous Form of Compound of Formula (I)

In one embodiment, there is provided compound of formula (I) inanhydrous form. In particular, there is provided compound of formula (I)in solid crystalline anhydrous form, obtained by crystallizing compoundof formula (I) from 1-propanol. Suitably, the 1-propanol is dry e.g.containing a maximum of around 0.9% w/w water. In one embodiment, the1-propanol has a maximum of 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or0.05% w/w of water. Suitably, the 1-propanol has maximum of 0.2% water.Suitably, crystallisation is performed in the presence of a metalscavenger. Suitable metal scavengers are materials that adsorb the metalwhile being easily separable from the compound of interest (i.e.compound of formula (I)). For example, functionalised silicas areparticularly useful as metal scavengers, as once the metal has beenadsorbed, the metal-silica complex may then be easily separated from thecompound of interest by filtration. Functional groups that form stablecomplexes with metal ions include groups containing one or more nitrogenand/or sulphur centres, and are well known to the person skilled in theart.

An example of a suitable commercially available metal scavenger isSiliaMetS® Thiol (a thiol-derivatised silica gel suitable for scavenginga variety of metals including Pd, Pt, Cu, Ag and Pb). Suitably, themetal scavenger is present in an amount sufficient to ensure that theresulting metal ion concentration is below 20 ppm, preferably below 10ppm. In one embodiment, the metal scavenger is present at 1-10% w/w, forexample 2-8% w/w or 5% w/w based on weight of compound of formula (I).Suitably crystallisation is performed by cooling the solution ofcompound of formula (I) and solvent from elevated temperature (e.g.80-95° C.), continuously (i.e. continuous cooling) or in stages (i.e.alternating between cooling and holding solution at a particulartemperature). Suitable temperature gradients (continuous or separate)for cooling include 95-15° C., 95-20° C., 90-20° C., 80-20° C. 95-90°C., 95-85° C., 95-80° C. 90-85° C. and 80-20° C. In one embodiment, thesolution is cooled from around 80-95° C. to ambient temperature (e.g.around 20-22° C.). The detailed preparation of such a solid crystallineanhydrous form of compound of formula (I) is provided in Example 2.Crystals of compound of formula (I) in solid crystalline form may becollected by usual separation techniques (e.g. by filtration orcentrifugation).

In one embodiment, there is provided a solid crystalline anhydrous formof compound of formula (I) having an XRPD pattern substantially as shownin FIG. 1. The method of obtaining the XRPD data is described in theGeneral Procedures and the data discussed in Example 3.

Thus, there is provided compound of formula (I) in a crystallineanhydrous form having an X-ray powder diffraction pattern with at leastone (for example, one, two, three, four, five, six, seven, eight, nineor all ten) peaks at 5.6, 7.9, 11.2, 12.3, 15.6, 17.6, 18.4, 21.4, 22.5,24.2 (±0.2 degrees, 2-theta values), these peaks being characteristic ofthe crystalline anhydrous form. The peaks at 17.6, 18.4, 22.5 and 24.2are particularly characteristic for the anhydrous form and therefore itis preferred to see at least one (for example one, two, three or allfour) of these peaks.

The chemical compatibility of the anhydrous form of compound(I) withlactose was investigated.

In order to assess chemical compatibility, compositions of the anhydrousform of compound of formula (I) with lactose were analysed by HPLC. Theresults are summarised in Example 4 where it is indicated that undercertain conditions the composition of anhydrous form and lactoseunderwent degradation. The degradation products were investigated andthe main degradant was identified by mass spectrometry as being one orboth of the two substances shown as D019328:

This degradation product is likely to be the result of the addition ofwater across the alkyne triple bond and may exist as one of two formswith identical mass (or may exist in both forms), depending on theorientation of the addition of the water across the triple bond. Thesame degradant has been observed during the forced degradation of theanhydrous form of compound of formula (I) with metal ions. As a resultof further studies, it appears that the degradation of the anhydrousform of compound of formula (I) requires metal ions and water and isaccelerated by elevated temperature.

Further investigation involving accelerated stability testing (i.e.exposure of the drug substance to 80° C. in a closed vial, see Example7) has led the inventors to confirm that at least the degradationproduct shown as D019492 in Scheme 1 (below) is generated. Moreover theinventors also concluded that a further degradation product (D019493)can result from the hydrolytic cleavage of the pyrimidinone ring andsubsequent intramolecular reaction with the alkyne group. D019349 is apresumed intermediary degradation product which was observed in certaincircumstances of temperature and RH in stability testing (data notshown).

The addition of magnesium stearate to the combination of anhydrous formof compound of formula (I) and lactose was investigated. The combinationof anhydrous form of compound of formula (I) with lactose and magnesiumstearate was found to be physically stable (Example 5). However,surprisingly, it was found that the addition of magnesium stearatecaused an increase in the chemical stability of the combination ofanhydrous form of compound of formula (I) and lactose (Example 6). Asimilar stabilising effect was found using other metal salts of stearicacid, specifically sodium stearate and calcium stearate (Example 7).

Without wishing to be bound by theory, it appears that the metal salt ofstearic acid such as magnesium stearate (or, it is believed, a metalsalt of stearyl fumarate) can act as a protecting agent against chemicaldegradation of the alkyne group in the compound of formula (I) andagainst chemical degradation of the pyrimidinone ring in the compound offormula (I) which is observed when the anhydrous form of compound offormula (I) is in a mixture with lactose.

Particulate Lactose as Carrier

As used herein, the term “lactose” refers to a lactose-containingcomponent, including α-lactose monohydrate, β-lactose monohydrate,α-lactose anhydrous, β-lactose anhydrous and amorphous lactose. Lactosecomponents may be processed by micronization, sieving, milling,compression, agglomeration or spray drying. Commercially available formsof lactose in various forms are also encompassed, for example Lactohale®(inhalation grade lactose; Frieslandfoods), InhaLac®70 (sieved lactosefor dry powder inhaler; Meggle) and Respitose® (sieved inhalation gradelactose; DFE Pharma) products. In one embodiment, the lactose componentis selected from the group consisting of α-lactose monohydrate,α-lactose anhydrous and amorphous lactose. Preferably, the lactose isα-lactose monohydrate.

In order to penetrate sufficiently far into the lungs, the particulateactive ingredient (in this case compound (I)) must be a suitable size asdescribed above. These small particles will have a tendency toagglomerate. The use of a carrier such as lactose prevents thisagglomeration and can improve flowability. Furthermore, the use of acarrier ensures that a correct and consistent dosage reaches the lungs.The active ingredient will usually form a monolayer on the largerlactose particle, then during inhalation the active ingredient and thecarrier are separated and the active ingredient is inhaled, while themajority of the carrier is not. As such, the use of particulate lactoseas a carrier for the active ingredient ensures that each dose of the drypowder pharmaceutical formulation releases the same amount of the activeingredient.

Generally, to prevent agglomeration of the small active particles,lactose with a particle size of approximately or at least ten times thatof the active ingredient is used (e.g. lactose having a D50approximately or at least ten times that of the active ingredient isused).

In one embodiment, the dry powder formulation of the present inventioncomprises particulate lactose having D50 in the range 40-150 μm.

The dry powder pharmaceutical formulation of the present inventioncomprises particulate lactose as carrier in an amount sufficient toensure that the correct and consistent dosage of the active ingredientreaches the lungs. In one embodiment, the dry powder pharmaceuticalformulation comprises from about 40 wt. % to about 99.88 wt. %, forexample from about 50 wt. % to about 99.88 wt. %, for example from about65 wt. % to about 99.88 wt. %, for example from about 75 wt. % to about99.99 wt. % of particulate lactose based on the weight of the dry powderpharmaceutical formulation. Preferably, the dry powder pharmaceuticalformulation comprises from about 80 wt. % to about 99.98 wt. % or forexample from about 80 wt % to about 99.9% wt %, for example from about85 wt. % to about 99.98 wt. %, for example from about 95 wt. % to about99 wt. % of particulate lactose based on the weight of the dry powderpharmaceutical composition.

Particulate Metal Salt of Stearic Acid Such as Magnesium Stearate orMetal Salt of Stearyl Fumarate as Stabilizing Agent

An example metal salt of stearic acid is magnesium stearate.

Alternative metal salts of stearic acid that may be employed includesalts of stearic acid formed with Group I and other Group II metals,such as sodium stearate, calcium stearate and lithium stearate. Othermetal salts of stearic acid that may be mentioned include zinc stearateand aluminium stearate.

Metal salts of stearyl fumarate (e.g. sodium stearyl fumarate) appear tohave similar properties to those of metal salts of stearic acid (seeShah et al, Drug development and Industrial pharmacy 1986, Vol. 12 No.8-9 , 1329-1346). In the inventors' opinion they can be employed as analternative to metal salts of stearic acid in the present invention.

As used herein the term “magnesium stearate” includes magnesium stearatetrihydrate, magnesium stearate dihydrate, magnesium stearate monohydrateand amorphous magnesium stearate. Magnesium stearate as defined hereinincludes a tolerance wherein any material defined as “magnesiumstearate” may contain up to 25% (e.g. up to 10% e.g. up to 5% e.g. up to1%) of palmitate salt.

More generally, metal salts of stearic acid or metal salts of stearylfumarate may be employed in anhydrous form or as a hydrate and maycontain up to 25% (e.g. up to 10% e.g. up to 5% e.g. up to 1%) ofpalmitate salt.

As used herein the expression “stabilizing agent selected from metalsalts of stearic acid such as magnesium stearate and metal salts ofstearyl fumarate” can include a mixture of metal salts of stearic acidand/or stearyl fumarate, although use of a single salt would bepreferred.

The metal salt of stearic acid such as magnesium stearate or metal saltof stearyl fumarate is typically obtained as a fine powder which neednot be micronized. Suitably the D50 of the metal salt of stearic acidsuch as magnesium stearate or the metal salt of stearyl fumarate isgreater than 5 μm e.g. around 10 μm or greater than 10 μm e.g. in therange 5 to 100 μm e.g. 5 to 50 μm e.g. 5 to 20 μm e.g. 10 to 20 μm.Magnesium stearate may for example be obtained from Avantor (Hyqual 2257brand) or Peter Greven. Sodium stearate and calcium stearate may, forexample, be obtained from Sigma-Aldrich. Sodium stearyl fumarate may,for example, be obtained from ScienceLab.

The dry powder pharmaceutical formulation of the present inventioncomprises particulate stabilizing agent selected from metal salt ofstearic acid such as magnesium stearate and metal salts of stearylfumarate in an amount sufficient to ensure the chemical stability of theformulation (“a stabilising amount”). Chemical stability is, forexample, demonstrated when the production of degradant D019328 (one orboth substances) is at a level of less than 0.2% wt. % following storageof the composition containing Compound of formula (I) for 4 weeks at 50°C. Alternatively or in addition, chemical stability is, for example,demonstrated when the production of degradant D019493 is at a level ofless than 0.5% wt. % following storage of the composition containingcompound of formula (I) for 2 weeks at 80° C. Alternatively, or inaddition, chemical stability is, for example, demonstrated when theproduction of degradant D019492 is at a level of less than 0.4% wt. %following storage of the composition containing Compound of formula (I)for 2 weeks at 80° C. In one embodiment, the dry powder pharmaceuticalformulation comprises from about 0.01 wt. % to about 15 wt. %, forexample 0.1 wt. % to about 10 wt. %, 10 wt. %, 5 wt. %, 2 wt. % or 1 wt.% of particulate stabilizing agent selected from metal salt of stearicacid such as magnesium stearate and metal salts of stearyl fumaratebased on the weight of the dry powder pharmaceutical formulation.Preferably, the dry powder pharmaceutical formulation comprises fromabout 0.5 wt. % to about 5 wt. % e.g. 1-2% w/w of particulatestabilizing agent selected from metal salt of stearic acid such asmagnesium stearate and metal salts of stearyl fumarate based on theweight of the dry powder pharmaceutical composition. Suitably, thestabilizing agent selected from metal salt of stearic acid such asmagnesium stearate and metal salts of stearyl fumarate is present in anamount sufficient to ensure the physical stability of the formulation.Physical stability is, for example, demonstrated when the IR spectrumand XRPD pattern of the composition (especially in relation tocharacteristics peaks of Compound of formula (I)) are substantiallyunaltered following storage of the composition containing Compound offormula (I) for 4 weeks at 50° C.

In one embodiment, the dry powder pharmaceutical formulation forinhalation of the present invention comprises:

-   -   (i) from about 0.02 to 50 wt. %        6-(2((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)        methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide        or a pharmaceutically acceptable salt thereof, including all        stereoisomers, tautomers and isotopic derivatives thereof and        solvates thereof in particulate form as active ingredient;    -   (ii) from about 40 wt. % to about 99.88 wt. % particulate        lactose; and    -   (iii) from about 0.1 wt. % to about 10 wt. % particulate        stabilizing agent selected from metal salts of stearic acid        (such as magnesium stearate) and metal salts of stearyl        fumarate.

In a further embodiment, the dry powder pharmaceutical formulation forinhalation of the present invention comprises:

-   -   (i) from about 0.02 to 50 wt. %        6-(2((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)        methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide        in free base form;    -   (ii) from about 40 wt. % to about 99.88 wt. % particulate        lactose; and    -   (iii) from about 0.1 wt. % to about 10 wt. % particulate        stabilizing agent selected from metal salts of stearic acid        (such as magnesium stearate) and metal salts of stearyl        fumarate.

A further aspect of the invention relates to the use of a stabilizingagent selected from metal salt of stearic acid such as magnesiumstearate and metal salts of stearyl fumarate in a pharmaceuticalformulation containing a compound of formula (I) and lactose to increasethe stability of the compound of formula (I) to chemical degradation(particularly in respect of metal ion catalysed addition of water to thealkyne group and/or hydrolysis of the pyrimidinone ring of the compoundof formula (I)) and to a method of increasing the stability of apharmaceutical formulation containing a compound of formula (I) andlactose to chemical degradation (particularly in respect of metal ioncatalysed addition of water to the alkyne group and/or hydrolysis of thepyrimidinone ring of the compound of formula (I)) which comprisesincluding in said formulation a stabilizing amount of a stabilizingagent selected from metal salts of stearic acid such as magnesiumstearate and metal salts of stearyl fumarate. Suitably the compound offormula (I) is in solid crystalline anhydrous form.

The preferred stabilizing agent is magnesium stearate.

Pharmaceutical Uses and Methods of Administration

There is provided according to one aspect of the present invention useof pharmaceutical formulation of the invention as a PI3 kinaseinhibitor.

In one embodiment there is provided the use of a pharmaceuticalformulation of the invention for the treatment of COPD and/or asthma, inparticular COPD or severe asthma, by inhalation i.e. by topicaladministration to the lung. Advantageously, administration to the lungallows the beneficial effects of the compounds to be realised whilstminimising the side-effects, for patients.

In one embodiment the pharmaceutical formulation of the invention issuitable for sensitizing patients to treatment with a corticosteroid.

The pharmaceutical formulations may conveniently be administered in unitdosage form and may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985).

Topical administration to the lung is achieved by use of an inhalationdevice.

Thus, an aspect of the invention includes an inhalation devicecomprising one or more doses of a pharmaceutical formulation accordingto the invention. Inhalation devices for dry powder formulations aretypically breath operated such that the dose is withdrawn from thedevice and administered to the subject using the power of the subject'slungs by inhaling from a mouthpiece. However, optionally, externalenergy may be provided to assist the administration of the dose.Typically the inhalation device will comprise a plurality of doses of apharmaceutical formulation according to the invention, e.g. 2 or 4 or 8or 28 or 30 or 60 or more doses. Thus the inhalation device may comprisea month's supply of doses. Optionally the doses are divided e.g. suchthat a dose is administered using two (or more) inhalations from theinhalation device. According to one embodiment of the invention thedoses of formulation are pre-metered in the inhalation device. Forexample the pre-metered doses may be contained in the pouches of ablister strip or disk or within capsules. In an embodiment, a dose ismetered into a capsule for use one by one in an inhalation deviceadapted to deliver the contents of a capsule to a subject uponinhalation. According to another embodiment of the invention the dosesare metered in use. Thus the inhalation device contains a reservoir ofdry powder and the device meters a dose of powder (typically on a fixedvolume basis) prior to or at the time of administration.

Example dry powder inhalation devices include SPINHALER, ECLIPSE,ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER, BREEZHALER/NEOHALER,FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN, ELPENHALER, DISKHALER,TURBUHALER, MIATHALER, TWISTHALER, NOVOLIZER, DISKUS, SKYEHALER, ORIELdry powder inhaler, MICRODOSE, ACCUHALER, PULVINAL, EASYHALER,ULTRAHALER, TAIFUN, PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX,XCELOVAIR, PROHALER and CLICKHALER. Another example is MONODOSE inhaler.

Optionally the inhalation device may be over-wrapped for storage toprotect against ingress of moisture. A desiccant may optionally beemployed within an over-wrap or within the device. Suitably thepharmaceutical formulation according to the invention in the inhalationdevice is protected from light.

The pharmaceutical formulations according to the invention may also beuseful in the treatment of respiratory disorders including COPD, chronicbronchitis, emphysema), asthma, paediatric asthma, cystic fibrosis,sarcoidosis and idiopathic pulmonary fibrosis and especially asthma,chronic bronchitis and COPD.

The pharmaceutical formulations according to the invention may comprisecompound of formula (I) as the sole active ingredient, or may compriseadditional active ingredients, e.g. active ingredients suitable fortreating the above mentioned conditions. For example possiblecombinations for treatment of respiratory disorders include combinationswith steroids (e.g. budesonide, beclomethasone dipropionate, fluticasonepropionate, mometasone furoate, fluticasone furoate, flunisolide,ciclesonide, triamcinolone), beta agonists (e.g. terbutaline,bambuterol, salbutamol, levalbuterol, salmeterol, formoterol,clenbuterol, fenoterol, broxaterol, indacaterol, reproterol, procaterol,vilanterol) and/or xanthines (e.g. theophylline), muscarinicantagonists, (e.g. ipratropium, tiotropium, oxitropium, glycopyrronium,glycopyrrolate, aclidinium, trospium), leukotriene antagonists (e.g.zafirlukast, pranlukast, zileuton, montelukast) and/or a p38 MAP kinaseinhibitor. It will be understood that any of the aforementioned activeingredients may be employed in the form of a pharmaceutically acceptablesalt.

In one embodiment, the pharmaceutical formulation of the invention isadministered in combination with an antiviral agent, for exampleacyclovir, oseltamivir (Tamiflu®), zanamivir (Relenza®) or interferon.

In one embodiment the combination of compound of formula (I) and otheractive ingredient(s) are co-formulated in the pharmaceutical formulationof the invention. In another embodiment, the other active ingredient(s)are administered in one or more separate pharmaceutical formulations.

In one embodiment compound of formula (I) is co-formulated in thepharmaceutical formulation of the invention or co-administered in aseparate formulation with a corticosteroid, for example for use inmaintenance therapy of asthma, COPD or lung cancer including preventionof the latter.

In one embodiment the pharmaceutical formulation of the invention isadministered by inhalation and a corticosteroid is administered orallyor by inhalation either in combination or separately.

The pharmaceutical formulation of the invention may also re-sensitisethe patient's condition to treatment with a corticosteroid, whenpreviously the patient's condition had become refractory to the same.

In one embodiment of the invention a dose of the pharmaceuticalformulation employed is equal to that suitable for use as a monotherapybut administered in combination with a corticosteroid.

In one embodiment a dose of the pharmaceutical formulation which wouldbe sub-therapeutic as a single agent is employed, and is administered incombination with a corticosteroid, thereby restoring patientresponsiveness to the latter, in instances where the patient hadpreviously become refractory to the same.

Additionally, the pharmaceutical formulation of the invention mayexhibit anti-viral activity and prove useful in the treatment of viralexacerbations of inflammatory conditions such as asthma and/or COPD.

The pharmaceutical formulation of the present invention may also beuseful in the prophylaxis, treatment or amelioration of influenza virus,rhinovirus and/or respiratory syncytial virus.

In one embodiment the presently disclosed pharmaceutical formulationsare useful in the treatment or prevention of cancer, in particular lungcancer, especially by topical administration to the lung.

Thus, in a further aspect, the present invention provides apharmaceutical formulation as described herein for use in the treatmentof one or more of the above mentioned conditions.

In a further aspect, the present invention provides a pharmaceuticalformulation as described herein for the manufacture of a medicament forthe treatment of one or more of the above mentioned conditions.

In a further aspect, the present invention provides a method oftreatment of the above mentioned conditions which comprisesadministering to a subject an effective amount of a pharmaceuticalformulation of the invention thereof.

Pharmaceutical formulations described herein may also be used in themanufacture of a medicament for the treatment of one or more of theabove-identified diseases.

The word “treatment” is intended to embrace prophylaxis as well astherapeutic treatment.

Unless otherwise specified, % values as used herein are % values byweight (wt. %).

Pharmaceutical formulations of the invention may have the advantage thatthey have improved physical stability (e.g. as measured by XRPD and/orIR analysis), improved chemical stability (e.g. as measured by HPLC),improved physical compatibility with lactose, improved chemicalcompatibility with lactose, improved particle size distribution onadministration (such as evidenced by improved fine particle mass) or mayhave other favourable properties as compared with similar formulationsthat do not contain a stabilizing agent selected from metal salt ofstearic acid such as magnesium stearate and metal salts of stearylfumarate.

Abbreviations

aq aqueousCOPD chronic obstructive pulmonary diseased doubletDCM dichloromethaneDMAP 4-dimethylaminopyridineDMSO dimethyl sulfoxideDPI dry powder inhalerDSC differential scanning calorimetryDVS dynamic vapour sorptionEDC.HCl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(ES⁺) electrospray ionization, positive modeEtOAc ethyl acetateHPLC high performance liquid chromatographyHPLC-MS high performance liquid chromatography mass spectrometryhr hour(s)IR infraredLPS lipopolysaccharide(M+H)⁺ protonated molecular ionMDI metered dose inhalerMeOH methanolMEK methylethylketoneMHz megahertzmin minute(s)

mm Millimetre(s)

ms mass spectrometrymTOR mammalian target of rapamycinm/z mass-to-charge ratioNH₄OAc ammonium acetateNMR nuclear magnetic resonance (spectroscopy)Pd(dppf)Cl₂ 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)ppm parts per millionq quartetquin quintetRH relative humidityRRT relative retention timeR^(t) retention timeRT room temperatures singlett tripletTBDMSCI tert-butyldimethylsilyl chlorideTGA thermogravimetric analysisTNFα tumour necrosis factor alphaXRPD X-ray powder diffraction

EXAMPLES General Procedures HPLC-MS

Performed on Agilent HP1200 systems using Agilent Extend C18 columns,(1.8 μm, 4.6×30 mm) at 40° C. and a flow rate of 2.5-4.5 mL min⁻¹,eluting with a H₂O-MeCN gradient containing 0.1% v/v formic acid over 4min. Gradient information: 0-3.00 min, ramped from 95% H₂O-5% MeCN to 5%H₂O-95% MeCN; 3.00-3.01 min, held at 5% H₂O-95% MeCN, flow rateincreased to 4.5 mL min⁻¹; 3.01-3.50 min, held at 5% H₂O-95% MeCN;3.50-3.60 min, returned to 95% H₂O-5% MeCN; flow rate reduced to 3.50 mLmin⁻¹; 3.60-3.90 min, held at 95% H₂O-5% MeCN; 3.90-4.00 min, held at95% H₂O-5% MeCN, flow rate reduced to 2.5 mL min⁻¹. UV detection wasperformed at 254 nm using an Agilent G1314B variable wavelengthdetector.

Mass Spectra (MS)

Obtained using electrospray ionization (ESI) over the range m/z 60 to2000 at a sampling rate of 1.6 sec/cycle using an Agilent G1956B, overm/z 150 to 850 at a sampling rate of 2 Hz using a Waters ZMD or over m/z100 to 1000 at a sampling rate of 2 Hz using a Shimadzu 2010 LC-MSsystem.

NMR spectra

¹H NMR spectra (except those of Example 7) were acquired on a BrukerAvance III spectrometer at 400 MHz using residual undeuterated solventas reference.

The ¹H NMR spectrum for Example 7 was acquired on a Bruker Avancespectrometer at 600 MHz using residual undeuterated solvent asreference.

X-Ray Powder Diffraction (XRPD)

XRPD patterns were acquired on a PANalytical (Philips) X′PertPRO MPDdiffractometer equipped with a Cu LFF X-ray tube (45 kV; 40 mA;Bragg-Brentano; spinner stage) were acquired using Cu Kα radiation andthe following measurement conditions:

scan mode: continuousscan range: 3 to 50° 2θstep size: 0.02°/stepcounting time: 30 sec/stepspinner revolution time: 1 secradiation type: CuKα

Incident Beam Path

program. divergence slit: 15 mmSoller slit: 0.04 radbeam mask: 15 mmanti scatter slit: 1°beam knife: +

Diffracted Beam Path

long anti scatter shield: +Soller slit: 0.04 radNi filter: +detector: X′Celerator

Samples were prepared by spreading on a zero background sample holder.

Infrared Spectrometry (IR)

Micro Attenuated Total Reflectance (microATR) was used and the samplewas analyzed using a suitable microATR accessory and the followingmeasurement conditions:

apparatus: Thermo Nexus 670 FTIR spectrometernumber of scans: 32resolution: 1 cm⁻¹wavelength range: 4000 to 400 cm⁻¹detector: DTGS with KBr windowsbeamsplitter: Ge on KBrmicro ATR accessory: Harrick Split Pea with Si crystal

Chemical Stability—High Performance Liquid Chromatography (HPLC)

HPLC analysis was carried out using the following operating conditions:

-   Column Waters Xbridge C18 (150×3.0×3.5 mm) or equivalent (a column    is considered equivalent if performance as specified in SST is met    and a comparable separation of all relevant compounds is    demonstrated).-   Column temperature 35° C.-   Sample temperature 10° C.-   Flow rate 0.45 ml/min-   Injection volume The injection volume can be adjusted as long as the    qualification limits of the system are not exceeded (detector and    injector) and the peak shape of the main compound is acceptable. As    a guide, 30 μl is considered suitable.-   Detection UV detection at 255 nm-   Mobile phase Preparation and composition:    -   A 10 mM ammonium acetate (0.771 g/l)+0.1%, v/v trifluoroacetic        acid in water    -   B Acetonitrile-   Gradient Analytical run time is 41 minutes

Time (minutes) Solvent 0 35 36 41 42 48 % A 95 30 0 0 95 95 % B 5 70 100100 5 5

With this HPLC method the degradant D019492 elutes at RRT0.86.

Chemical Stability—Ultra High Pressure Liquid Chromatography (UPLC)

UPLC analysis was carried out using the following operating conditions:

-   Column Acquity BEH C₁₈; 2.1×150 mm; 1.7 μm or equivalent (a column    is considered equivalent if performance as specified in SST is met    and a comparable separation of all relevant compounds is    demonstrated)-   Column temperature 35° C.-   Sample temperature 10° C.-   Flow rate 0.40 ml/min-   Injection volume The injection volume can be adjusted as long as the    qualification limits of the system are not exceeded (detector and    injector) and the peak shape of the main compound is acceptable. As    a guide, 4 μl is considered suitable.-   Detection UV detection at 255 nm-   Mobile phase Preparation and composition:    -   A 10 mM ammonium acetate (0.771 g/l)+0.1%, v/v trifluoroacetic        acid in water    -   B Acetonitrile-   Gradient Analytical run time is 23 minutes

Time (minutes) Solvent 0 19 20 23 23.5 28 % A 95 30 0 0 95 95 % B 5 70100 100 5 5

With this UPLC method the degradant D019492 elutes at RRT=0.92-0.93 andthe degradant D019493 elutes at RRT=0.86-0.87.

Reagents and Suppliers

Lactohale200®: supplied by Frieslandfoods. Particle size (Sympatec):D10: 5-15 μm; D50: 50-100 μm; D90: 120-160 μm.Magnesium stearate: Grade Hyqual® 2257; supplied by Avantor. Particlesize: D10: typically 3 μm; D50: typically 11.5 μm (10.5-16.5 μm); D90:typically 24 μm (18-28 μm). Supplied as a fine powder.

Example 1 Preparation of6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide5-Bromo-3-(2-chlorobenzyl)-2-(chloromethyl)quinazolin-4(3H)-one (2)

To a stirred solution of 2-amino-6-bromo-benzoic acid (3.06 g, 14.2mmol) in toluene (75 mL) cooled to 0° C. in an ice-bath was addedpyridine (0.60 mL, 7.10 mmol) followed by a solution of chloroacetylchloride (2.26 mL, 28.4 mmol) in toluene (75 mL) drop-wise over 1 hr.The reaction mixture was allowed to warm to RT, and was heated at 115°C. for 3 hr and then allowed to cool to RT. The solvent volume wasreduced by half by evaporation in vacuo. Upon standing overnight, theproduct precipitated and was collected by filtration to afford2-bromo-6-(2-chloroacetamido)benzoic acid (1a, X═Cl) (1.44 g) as a whitesolid: m/z 290/292 (M+H)⁺ (ES⁺). The filtrate was concentrated in vacuoand the residue triturated with ethanol/heptane to afford2-bromo-6-(2-hydroxyacetamido) benzoic acid (1b X═OH) (1.02 g, combinedyield, 59%): m/z 274/276 (M+H)⁺ (ES⁺). Both 1a and 1 b can be usedwithout further purification in the next step.

To a stirred mixture of compound (1a) (7.50 g, 27.4 mmol),2-chlorobenzylamine (5.00 mL, 41.05 mmol) and triethylamine (5.70 mL,41.1 mmol) in toluene (250 mL) was added a solution of phosphorustrichloride (2.60 mL, 30.1 mmol) in toluene (250 mL) dropwise over 1 hr.The reaction mixture was heated to 110° C. for 24 hr, whereupon the hotsolution was decanted and concentrated in vacuo. The residue wastriturated with propan-2-ol (50 mL) to afford the title compound (2)(6.41 g, 59%) as a yellow solid: R^(t) 2.67 min; m/z 397/399 (M+H)⁺(ES⁺).

3-(3-(tert-Butyldimethylsilyloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(6)

To a stirred suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine(3) (8.22 g, 31.5 mmol), 3-phenol boronic acid (13.0 g, 94.5 mmol) andpotassium phosphate (10.0 g, 47.3 mmol) in degassed DMF/water (3:2, 140mL) was added Pd(dppf)Cl₂ (13.0 g, 15.7 mmol). The reaction mixture wasflushed with nitrogen, heated at 120° C. for 2 hr and then allowed tocool to RT. The reaction mixture was diluted with EtOAc (500 mL) andhydrochloric acid (2 M, 500 mL) and the resulting suspension wasfiltered. The filtrate was extracted with hydrochloric acid (2 M, 2×500mL). The combined aqueous extracts were basified with a saturatedaqueous solution of sodium carbonate to pH 10. The precipitate formedwas filtered and the filtrate was extracted with EtOAc (3×1 L). Thecombined organic extracts were dried, filtered and the solvent removedin vacuo to afford a grey solid. All solid materials generated duringthe workup procedure were combined and triturated with DCM to afford3-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (5) (6.04 g, 84%) asa grey solid: m/z 228 (M+H)⁺ (ES⁺).

To a stirred solution of the phenol (5) (4.69 g, 20.66 mmol) andimidazole (2.10 g, 30.99 mmol) in dry DMF (100 mL) was added TBDMSCI(4.70 g, 30.99 mmol). After 16 hr, further aliquots of imidazole (2.10g, 30.99 mmol) and TBDMSCI (4.70 g, 30.99 mmol) were added and themixture was stirred for 48 hr. The reaction mixture was diluted withwater (120 mL) and extracted with DCM (2×200 mL). The combined organicextracts were washed with water (2×200 mL), dried, filtered and thevolume reduced to approximately 100 mL by evaporation in vacuo. Theresulting slurry was filtered and the solid washed with heptane (50 mL)to afford the title compound (6) (6.05 g, 85%) as an off-white solid:m/z 343 (M+H)⁺ (ES⁺).

Intermediate A:2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-bromo-3-(2-chlorobenzyl)quinazolin-4(3H)-one

To a stirred mixture of5-bromo-3-(2-chlorobenzyl)-2-(chloromethyl)quinazolin-4(3H)-one (2) (100mg, 0.25 mmol) and potassium carbonate (42 mg, 0.30 mmol) in DMF (2.5mL) was added a solution of3-(3-(tert-butyldimethylsilyloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(6) (94 mg, 0.28 mmol) in DMF (2.5 mL) and the reaction mixture wasstirred at RT for 18 hr. Potassium carbonate (3×35 mg, 0.75 mmol) wasadded in three portions over 30 hr. after which the solvent was removedin vacuo and the crude material was purified by flash columnchromatography, eluting with 4.5% methanol in DCM, to afford the titlecompound, Intermediate A, (94 mg, 64%) as a off-white solid: R^(t) 2.01min; m/z 588/590 (M+H)⁺, (ES⁺).

Intermediate B: N,N-bis(2-Methoxyethyl)hex-5-ynamide

To a solution of hex-5-ynoic acid (7.11 g, 63.4 mmol), EDC.HCl (14.0 g,72.9 mmol) and DMAP (387 mg, 3.17 mmol) in DCM (600 mL) at 0° C. wasadded bis(2-methoxyethyl)amine (9.3 mL, 63 mmol). The resulting mixturewas warmed to RT for 20 hr and was then washed with hydrochloric acid (1M, 2×500 mL) and with water (500 mL). The organic layer was dried andwas evaporated in vacuo to afford the title compound, Intermediate B, asa yellow oil (16 g, 97%): ¹H NMR (400 MHz, CDCl₃) δ: 1.88 (3H, m), 2.26(2H, m), 2.49 (2H, m), 3.32 (6H, s), 3.51 (4H, m), 3.55 (4H, m)

6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide(I)

Intermediate A((2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-bromo-3-(2-chlorobenzyl)quinazolin-4(3H)-one(65.7 g, 1.0 eq.)), copper(I) iodide (1.06 g, 0.05 moles/mol),bis(triphenylphosphine)palladium(II) chloride (3.92 g, 0.05 moles/mol),Intermediate B (N,N-bis(2-methoxyethyl)hex-5-ynamide (63.42 g, 2.5moles/mol) and diethylamine (837.05 mL; 591.21 g, 7.5 L/mol) were addedto a 2 L reactor and the mixture degassed with argon purging. Thereaction mixture was warmed to 55° C. (reflux temperature) over 30minutes and then stirred at 55° C. After 2 hours the mixture was cooledto 22° C. before being concentrated in vacuo to produce a dark brownsemi solid residue (201.0 g). The residue was then dissolved in MEK(781mL) and water added (223 mL). After stirring strongly for 5 minutes thelayers were separated and the aqueous layer discarded. The organic layerwas washed with 10% w/v aqueous NH₄OAc (300 mL) and 2% w/v aqueous NaCl(112 mL) before being partly concentrated in vacuo to an heterogeneousmixture in MEK (230 g). The mixture was stirred for 16 hours thenfiltered, and the precipitate was washed with MEK (3×25 mL). Theresulting solid was dried at 50° C. in vacuo for 18 hours to give“crude”6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide(compound of formula (I)) (54.13 g; 0.66 equiv; 65.97% yield).

Crude compound of formula (I) (53.5 g; 1.00 equiv), methanol (7.28 mL,0.1 L/mol) and dichloromethane (145.53 mL, 2 L/mol) were stirred in a250 mL reactor at 22° C. After 4 hours the solid was filtered and washedwith dichloromethane (29 mL) before being dried in vacuo at 40° C. for18 hours to obtain compound of formula (I) (the title compound) (45.3 g;0.85 equiv; 84.67% crystallization yield) as an off-white solid.

Example 2 Preparation of Compound of Formula (I) in Solid CrystallineAnhydrous Form

All reactions described within this example were carried out under aflow of nitrogen gas. Compound of formula (I) as prepared in Example 1(14.0 g) and 1-propanol (210 mL, 15 L/kg) were added to a 400 mLcrystallization vessel. The resulting heterogeneous mixture was stirredand warmed to 90° C. (with the mixture becoming homogeneous at 85° C.).Once the solution had reached 90° C., a metal scavenger (SiliaMetS®Thiol 0.7 g (5% w/w)) was added and the mixture warmed to 95° C. Afterstirring for 15 minutes at 95° C. the mixture was cooled to 90° C. andstirred for a further 2 hours at 90° C. The metal scavenger was thenfiltered and the homogeneous filtrate was again stirred and warmed to95° C., before being cooled to 85° C. and stirred for 8 hours. Thefiltrate was then cooled over 8 hours to 20° C. and stirred for anadditional 6 hours at 20° C. The product was then filtered and washedwith 1-propanol (6 mL) before being dried in vacuo at 50° C. for 18hours to afford compound of formula (I) in anhydrous form (12.6 g, 90%)as a white solid.

The above method may optionally be adapted to facilitate crystallizationwith seeding.

Example 3 XRPD Analysis of Compound of Formula (I) in Solid CrystallineAnhydrous Form

XRPD analysis of the anhydrous form of compound of formula (I) (Example2) was undertaken using the method described in General Procedures. Theresulting diffraction pattern is shown in FIG. 1. The XRPD patternshowed diffraction peaks without the presence of a halo, therebyindicating that both materials are crystalline. Characteristic peaks ofthe forms are given below in Table 1:

TABLE 1 Characteristic XRPD peaks for the anhydrous form of compound offormula (I) XRPD peaks (±0.2 degrees, 2-theta values) 5.6 7.9 11.2 12.315.6 17.6 18.4 21.4 22.5 24.2

Example 4 HPLC Analysis of Compound of Formula (I) in Solid CrystallineAnhydrous Form (Micronized) with Lactose

The chemical compatibility of the solid crystalline anhydrous form ofcompound of formula (I) (micronized) in combination with lactose wasdetermined by HPLC analysis.

Micronized anhydrous form of compound of formula (I) was prepared usinga jet mill micronization device (1.5 bar) (manufactured by HosokawaAlpine) to produce the following particle size distribution: D10=1.40μm; D50=2.77 μm and D90=5.29 μm (the particle size distribution wasdetermined using laser diffraction (Malvern Mastersizer instrument).

The test batch was taken from stock containing 3.519 mg anhydrous formof compound of formula (I) (micronized) and 6006.64 mg Lactohale200.

The mixtures were analysed by HPLC at time zero and after differentconditions of storage. Samples were stored under the followingconditions: (i) 1, 2, 3 and 4 weeks at 50° C. (ii) 1 week at 80° C.(iii) 1, 2, 3 and 4 weeks at 40° C./75% RH.

The data shown in Table 2 indicate that significant degradation wasobserved after storage for 1 week at 80° C. and degradation was alsoobserved after storage for 4 weeks at 50° C. These results suggest thatthe anhydrous form (micronized) of compound of formula (I) is notchemically stable in combination with lactose, therefore the twocomponents would not be compatible in a pharmaceutical formulation.

The peak at RRT 0.86 has been attributed to the hydrated derivative(s)D019328 shown above.

TABLE 2 stability data for the anhydrous form of the compound of formula(I) (micronized) with lactose RRT* RRT* RRT* RRT* RRT* Conditions 0.800.86 0.97 1.14 1.32 T = zero 0.21 0.12 0.12 0.13 1 week 50° C. 0.17 0.230.10 0.12 1 week 80° C. 0.52 2.53 0.78 0.19 0.12 1 week 40° C./75% RH0.19 0.12 0.11 0.13 2 weeks 50° C. 0.19 0.30 0.12 0.13 2 weeks 40°C./75% RH 0.17 0.11 0.12 0.13 3 weeks 50° C. 0.19 0.38 0.12 0.14 3 weeks40° C./75% RH 0.19 0.08 0.11 0.14 4 weeks 50° C. 0.19 0.54 0.11 0.13 4weeks 40° C./75% RH 0.18 0.20 0.11 0.14 *Area % by HPLC at the RRTindicated. Compound of formula (I) has RRT = 1.0

Example 5 XRPD/IR Analysis of Compound of Formula (I) in SolidCrystalline Anhydrous Form with Lactose and Magnesium Stearate

A mixture of the solid crystalline anhydrous form (micronized) ofcompound of formula (I) with lactose was prepared with the addition of1% magnesium stearate (micronization of compound of formula (I) asdescribed in Example 4).

Blend preparation: about 500 mg of Lactohale200® and about 10 mgmagnesium stearate were added to an agate mortar before being mixedusing a pestle and plastic blade (Feton) for 5 minutes. About 500 mg ofanhydrous compound of formula (I) (micronized) was added to the mixtureand the blend was mixed for a further 5 minutes.

The mixtures were stored under different temperatures and humidities andwere analysed by XRPD and IR at time zero and after 1 week and 4 weeksof storage. The conditions for 1 week storage were: 40° C./75% RH open;1 week 50° C. closed; and 1 week 80° C. closed. The conditions for 4week stability storage were: 4 weeks 50° C. closed; 4 weeks 40° C./75%RH open.

The IR spectrum acquired at time zero is shown in FIG. 2. IR spectrawere prepared for samples in the stability studies. No differences wereobserved between the IR spectra of the 1 and 4 week stability samplesand the IR spectrum at time zero. No interaction between the anhydrousform; lactose and magnesium stearate was observed and the anhydrous formremained stable under all storage conditions.

The XRPD spectrum acquired at time zero is shown in FIG. 3. XRPD spectrawere prepared for samples in the stability studies. The generated XRPDpatterns of the 1 and 4 week stability samples were similar to thediffraction pattern at time zero. It was clearly evident that thetypical diffraction peaks of the anhydrous form did not change in thepresence of Lactohale200® and magnesium stearate, indicating that theanhydrous form is physically stable in the presence of lactose andmagnesium stearate.

The IR spectra showed no interaction between the anhydrous form, thelactose and the magnesium stearate, and the XRPD results showed thatthere was no solid state conversion of the anhydrous form. As a result,it may be concluded that the anhydrous form is physically compatiblewith lactose and magnesium stearate.

Example 6 HPLC Analysis of Compound of Formula (I) in Anhydrous Formwith Lactose and Magnesium Stearate

The chemical compatibility of the solid crystalline anhydrous form(micronized) of compound of formula (I) in combination with lactose and1% magnesium stearate was determined by HPLC analysis (micronization ofcompound of formula (I) as described in Example 4).

The test batch was taken from stock containing 3.704 mg anhydrous formof compound of formula (I) (micronized), 6017.90 mg Lactohale200 and67.33 mg magnesium stearate.

The data shown in Table 3 indicate a significant increase in chemicalstability compared with the same composition with the absence ofmagnesium stearate (see Table 2), as evidenced by only a small amount ofdegradation observed after storage for 1 week at 80° C. (see e.g. RRT0.86, 0.28%). These results suggest that the chemical stability of theanhydrous form (micronized) of compound of formula (I) with lactose issignificantly improved by the addition of magnesium stearate to thecomposition. As such, the addition of magnesium stearate improves thechemical compatibility of the anhydrous form (micronized) of compound offormula (I) in combination with lactose such that they could becompatible in a pharmaceutical formulation.

TABLE 3 stability data for the anhydrous form of the compound of formula(I) (micronized) with lactose and magnesium stearate RRT* RRT* RRT* RRT*Conditions 0.80 0.86 1.14 1.32 T = zero 0.21 0.10 0.12 0.13 1 week 50°C. 0.20 0.11 0.11 0.13 1 week 80° C. 0.19 0.28 0.11 0.13 1 week 40°C./75% RH 0.20 0.11 0.11 0.13 2 weeks 50° C. 0.20 0.08 0.11 0.14 2 weeks40° C./75% RH 0.21 0.11 0.11 0.13 3 weeks 50° C. 0.20 0.13 0.11 0.13 3weeks 40° C./75% RH 0.20 0.11 0.11 0.14 4 weeks 50° C. 0.19 0.12 0.110.14 4 w 40° C./75% RH 0.20 0.10 0.10 0.13 *Area % by HPLC at the RRTindicated. Compound of formula (I) has RRT = 1.0

Example 7 UPLC Analysis of Compound of Formula (I) in Anhydrous Formwith Lactose and Metal Salts of Stearic Acid

The chemical compatibility of the solid crystalline anhydrous form(micronized) of compound of formula (I) in combination with lactose and1% metal salt of stearic acid (magnesium stearate, sodium stearate andcalcium stearate) was determined by UPLC analysis (micronization ofcompound of formula (I) as described in Example 4).

Test samples were prepared as described in Table 4 below:

TABLE 4 test samples for UPLC analysis after accelerated stabilitytesting solid crystalline anhydrous form Metal salt of (micronized)Lactohale 200 stearic acid of compound (I) sample 1/ sample 1/ Samplesample 1/sample 2 sample 2 sample 2 Drug only 0.50 mg/ 0.47 mg Drug and0.58 mg/ 749.84 mg/ lactose 0.47 mg 750.06 mg Drug, lactose, 0.46 mg/749.97 mg/ 7.40 mg/ Mg stearate 0.51 mg 751.59 mg 7.55 mg Drug, lactose,0.49 mg/ 751.08 mg/ 7.67 mg/ Ca stearate 0.45 mg 753.53 mg 7.80 mg Drug,lactose, 0.48 mg/ 750.20 mg/ 7.78 mg/ Na stearate 0.45 mg 750.42 mg 7.59mg

Samples were dispensed into vials, sealed with caps and kept at 80° C.for 1 or 2 weeks. Sample 1 was used for the 1 week studies and sample 2was used for the 2 week studies.

Results are shown in Table 5 below:

TABLE 5 results of UPLC analysis after accelerated stability testing 1week 1 week 2 weeks 2 weeks 80° C. 80° C. 80° C. 80° C. Sample RRT* 0.87RRT* 0.92 RRT* 0.87 RRT* 0.92 Drug only 0.00 0.08 0.00 0.08 Drug and0.58 0.39 1.80 0.77 lactose Drug, lactose, 0.28 0.29 0.06 0.18 Mgstearate Drug, lactose, 0.11 0.19 0.17 0.19 Ca stearate Drug, lactose,0.00 0.09 0.00 0.09 Na stearate *Area % by UPLC at RRT indicated.Compound of formula (I) has RRT = 1.0

Mass spectroscopy analysis indicates that the substance with RRT=0.87 isD019493 and the substance with RRT=0.92 is D019492 (confirmed by NMR)(see Scheme 1). The NMR resonance assignments for D019492 are given inTable 6:

TABLE 6 ¹H NMR resonance assignments for D019492 ¹H NMR assignments (600MHz, DMSO-d₆) δ ppm D019492 1.59 (quin, J = 7.30 Hz, 2 H) 2.20 (t, J =7.55 Hz, 2 H) 2.46-2.49 (m, 2 H) 3.18 (d, J = 7.90 Hz, 6 H) 3.29-3.39(m, 8 H) 4.23 (s, 2 H) 5.24 (s, 2 H) 5.76 (s, 2 H) 6.08 (d, J = 7.55 Hz,1 H) 6.75 (t, J = 7.55 Hz, 1 H) 6.83 (dd, J = 8.12, 1.70 Hz, 1 H) 6.90(d, J = 7.55 Hz, 1 H) 6.91-6.93 (m, 1 H) 7.01 (t, J = 7.55 Hz, 1 H) 7.09(d, J = 7.55 Hz, 1 H) 7.29 (m, J = 7.93, 7.93 Hz, 1 H) 7.32 (d, J = 7.18Hz, 1 H) 7.66 (d, J = 7.93 Hz, 1 H) 7.77-7.82 (m, 1 H) 8.17 (s, 1 H)9.67 (s, 1 H)

The data shown in Table 5 indicate a significant increase in chemicalstability for formulations containing a metal salt of stearic acidcompared with the same composition in the absence of a metal salt ofstearic acid, as evidenced by a comparatively small amount ofdegradation observed after storage for 1 or 2 weeks at 80° C. Theseresults suggest that the chemical stability of the anhydrous form ofcompound of formula (I) with lactose is significantly improved by theaddition of metal salts of stearic acid to the composition. Therefore,the addition of metal salts of stearic acid improves the chemicalcompatibility of the anhydrous form of compound of formula (I) incombination with lactose such that they could be compatible in apharmaceutical formulation.

Example 8 Preparation of Pharmaceutical Formulations According to theInvention

An exemplary pharmaceutical formulation of the invention consists of 0.5wt. % of compound of formula (I) (solid crystalline anhydrous form,micronised), 98.5 wt. % lactose monohydrate (inhalation grade) and 1.0wt. % magnesium stearate, wherein the wt. % of all components is basedon the weight of the dry pharmaceutical formulation.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

1. A dry powder pharmaceutical formulation for inhalation comprising: acompound of formula (I)

that is6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamideor a pharmaceutically acceptable salt thereof, including allstereoisomers, tautomers and isotopic derivatives thereof and solvatesthereof in particulate form as active ingredient; (ii) particulatelactose as carrier; and (iii) a particulate stabilizing agent selectedfrom metal salts of stearic acid and metal salts of stearyl fumarate. 2.A pharmaceutical formulation according to claim 1, wherein the compoundof formula (I) is in its free base form.
 3. A pharmaceutical formulationaccording to claim 1, wherein the compound of formula (I) is in solidcrystalline form.
 4. A pharmaceutical formulation according to claim 3,wherein the compound of formula (I) is in anhydrous form.
 5. Apharmaceutical formulation according to claim 4, wherein the compound offormula (I) is in solid crystalline form having the X-ray powderdiffraction pattern substantially as shown in FIG.
 1. 6. Apharmaceutical formulation according to claim 4, wherein the compound offormula (I) is in solid crystalline form having a X-ray powderdiffraction pattern containing one, two, three or four peaks selectedfrom (±0.2) 17.6, 18.4, 22.5 and 24.2 degrees 2-theta.
 7. Apharmaceutical formulation according to claim 1 wherein the activeingredient has been micronized.
 8. A pharmaceutical formulationaccording to claim 1 wherein the stabilizing agent is a metal salt ofstearic acid.
 9. A pharmaceutical formulation according to claim 8wherein the stabilizing agent is magnesium stearate.
 10. Apharmaceutical formulation according to claim 1 wherein the lactose isα-lactose monohydrate.
 11. An inhalation device comprising one or moredoses of a pharmaceutical formulation according to claim
 1. 12.(canceled)
 13. (canceled)
 14. A method of treatment of a conditionselected from: COPD (including chronic bronchitis and emphysema), asthmaincluding paediatric asthma, cystic fibrosis, sarcoidosis, idiopathicpulmonary fibrosis, cachexia and inhibition of the growth and metastasisof lung tumours including non-small cell lung carcinoma which comprisesadministering to a subject an effective amount of a pharmaceuticalformulation according to claim
 1. 15-20. (canceled)
 21. A method ofincreasing the stability of a pharmaceutical formulation containing acompound of formula (I) and lactose to chemical degradation whichcomprises including in said formulation a stabilizing amount of astabilizing agent selected from metal salts of stearic acid such asmagnesium stearate and metal salts of stearyl fumarate.
 22. A methodaccording to claim 21 wherein the compound of formula (I) is in solidcrystalline anhydrous form.
 23. A method according to claim 22 whereinthe compound of formula (I) is in solid crystalline form having theX-ray powder diffraction pattern substantially as shown in FIG.
 1. 24. Amethod according to claim 22 wherein the compound of formula (I) is insolid crystalline form having the X-ray powder diffraction patterncontaining one, two, three or four peaks selected from (±0.2) 17.6,18.4, 22.5 and 24.2 degrees 2-theta.
 25. A method according to claim 21wherein the stabilizing agent is a metal salt of stearic acid.
 26. Amethod according to claim 25 wherein the stabilizing agent is magnesiumstearate.